Epigenetic modifications play an important role in regulating gene activity. In many cases, cancer can be regarded as an outcome of the misregulation of genes involved in cell division, differentiation, and development. Therefore, it is not surprising that some of the enzymes involved in epigenetic modifications have been linked to cancer. One such epigenetic modification is histone methylation. Of the histone methyltransferases (HMTases) identified so far, yeast Dot1 and its human counterpart, hDOTIL, are unique due to their lack of the SET domain, a signature HMTase motif, and their ability to methylate lysine 79 located within the globular domain of histone H3. Toward understanding the biological function of hDOTIL, we set out to identify its functional partners. Two proteins that we identified are AF10 and ENL, both of which are MLL (mix lineage leukemia) fusion partner involved in acute myeloid leukemia. Our preliminary studies indicate that hDOT1 L and its associated H3-K79 methyltransferase activity play an important role in MLL-AF10 mediated leukemic transformation. The evidence that support our conclusion include: 1) The OM-LZ domain of AF10, required for leukemogenesis of the MLL-AF10 fusion protein, is both necessary and sufficient in mediating the AF10-hDOT1L interaction; 2) Direct fusion of hDOTIL to MLL results in transformation of bone marrow progenitor cells and the transformation ability of MLL-hDOT1L depends on the HMTase activity of hDOTIL; 3) An HMTase deficient hDOTIL mutant can prevent proliferation of leukemia cells transformed by MLL-AF10; 4) Similar to that of MLL-AF10, MLL-hDOT1L transformed cells exhibit characteristic up-regulation of late Hoxa genes, including Hoxa7 and Hoxa9; 5) Up-regulation of Hoxa9 in MLL-AF10 transformed cells correlates with MLL-AF10 recruitment and H3-K79 hypermethylation. In addition, we have evidence that hDOTIL is also involved in CALM-AF10-mediated leukemia. Our studies indicate that mis-targeting of hDOTIL and subsequent up-regulation of leukemia relevant genes, such as Hoxa9, by MLL-AF10 or CALM-AF10 may play an important role in leukemogenesis. To extend these studies, we have established the following specific aims: 1. Evaluate the role of mDOTIL in MLL-AF10-mediated leukemogenesis in vivo. We will perform transformation assay in vivo and evaluate the leukemogenic capability of MLL-hDOT1L in mice. We will also use a siRNA knockdown approach to evaluate the role of mDOTIL in MLL-AF10-mediated transformation of mouse bone marrow progenitor cells. 2. Understand the molecular basis of leukemic transformation by MLL-hDOT1L. This will be accomplished by first determine the minimum sequence on hDOTIL that when fused to MLL is leukemogenic. Then, the role of the required sequences in mediating MLL dimerization or recruitment of other factors will be examined. 3. Evaluate the role of hDOTIL in CALM-AF10 mediated leukemogenesis. We will perform transformation assays in vivo and evaluate the leukemogenic capability of CALM-AF10 and CALM-hDOT1L in mice. We will also use a similar approach as in Aim 1 to evaluate the role of mDOTIL in CALM-AF10-mediated transformation of mouse bone marrow progenitor cells. 4. Understand the molecular basis of leukemic transformation by CALM-AF10. We have evidence that hDOTIL contributes to nuclear localization for CALM-AF10. We will map the nuclear localization signal of hDOTIL and the AF10 interaction domains. We will also study how mis-targeting of hDOTIL by CALM-AF10 results in Hox gene upregulation and leukemogenesis. [unreadable] [unreadable] [unreadable]